We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC 50 value of 0.24 mM, whereas thyroid hormone (triiodo-L-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC 50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC 50 (concentration that gives 50% activation) of 7 μM] shifts the equilibrium to the tetrameric active Rstate, which has a similar activity to that of the constitutively fully active isoform M1PYK. Proliferation assays using HCT-116 cells showed that addition of inhibitors phenylalanine and T3 both increased cell proliferation, whereas addition of the activator F16BP reduced proliferation. F16BP abrogates the inhibitory effect of both phenylalanine and T3, highlighting a dominant role of M2PYK allosteric activation in the regulation of cancer proliferation. X-ray structures show constitutively fully active M1PYK and F16BP-bound M2PYK in an R-state conformation with a lysine at the dimer-interface acting as a peg in a hole, locking the active tetramer conformation. Binding of phenylalanine in an allosteric pocket induces a 13°rotation of the protomers, destroying the peg-in-hole R-state interface. This distinct T-state tetramer is stabilized by flipped out Trp/Arg side chains that stack across the dimer interface. Xray structures and biophysical binding data of M2PYK complexes explain how, at a molecular level, fluctuations in concentrations of amino acids, thyroid hormone, and glucose metabolites switch M2PYK on and off to provide the cell with a nutrient sensing and growth signaling mechanism.allosteric regulation | nutrient sensor | thyroid hormone T3 | Warburg effect T he last of 10 enzymatic steps used to convert glucose to pyruvate is carried out by pyruvate kinase (PYK), which transfers a phosphate from phosphoenolpyruvate to ADP to generate ATP. There are four human PYK isoforms (1); RPYK is restricted to erythrocytes, LPYK is found predominantly in liver and kidney, M1PYK is in muscle and brain, and M2PYK is found in fetal tissues and in proliferating cells. All four isoforms are active as tetramers; M1PYK is constitutively fully active, whereas R-, L-, and M2PYKs are activated by the effector molecule fructose-1,6-bisphosphate (F16BP) (2). M2PYK is a splice variant of the nonallosteric M1PYK isoform and differs by 22 amino acid residues (3). Recent quantification of the concentrations of constitutively fully active M1PYK and allosterically regulated M2PYK isoforms in both cancerous and control tissue samples has revealed that M2PYK is almost always the most abundant isoform in cancer cells, although it can also be predominant in matched control tissues (4). The up-regulation of the M2PYK isoform plays a key role in cancer metabolism (3) and explains the Warburg effect, in which proliferating cancer cells metabolize increas...
Over the last decade, cross-linking/mass spectrometry (CLMS) has developed into a robust and flexible tool that provides medium-resolution structural information on previously intractable protein complexes and and their interactions. CLMS data describes the proximity of amino acid residues and therefore information on the fold of proteins, and the topology of their complexes. Here, we describe notable successes of this technique and common pipelines. Novel CLMS applications such as in-cell cross-linking, probing conformational changes and tertiary structure determination are beginning to make large contributions to molecular biology and the emerging fields of structural systems biology and interactomics.
Highlights d 3.9-Å cryo-EM structure of the cohesin complex in a DNA gripping state intermediate d DNA is trapped between two gates that lead into the cohesin ring d The kleisin N-tail guides DNA through a kleisin N-gate into the gripping state d ATP hydrolysis opens the head gate to complete DNA entry
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.